Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

Elemental iron protects gut microbiota against oxygen-induced dysbiosis.

Gut dysbiosis induced by oxygen and reactive oxygen species may be related to the development of inflammation, resulting in metabolic syndrome and associated-conditions in the gut. Here we show that elemental iron can serve as an antioxidant and reverse the oxygen-induced dysbiosis. Fecal samples from three healthy donors were fermented with elemental iron and/or oxygen. 16S rRNA analysis revealed that elemental iron reversed the oxygen-induced disruption of Shannon index diversity of the gut microbiota.The bacteria lacking enzymatic antioxidant systems also increased after iron treatment. Inter-individual differences, which corresponded to iron oxidation patterns, were observed for the tested donors. Gut bacteria responding to oxygen and iron treatments were identified as guilds, among which, Escherichia-Shigella was promoted by oxygen and depressed by elemental iron, while changes in bacteria such as Bifidobacterium, Blautia, Eubacterium, Ruminococcaceae, Flavonifractor, Oscillibacter, and Lachnospiraceae were reversed by elemental iron after oxygen treatment. Short-chain fatty acid production was inhibited by oxygen and this effect was partially reversed by elemental iron. These results suggested that elemental iron can regulate the oxygen/ROS state and protect the gut microbiota from oxidative stress.

Gut microbial signatures of patients with primary hepatocellular carcinoma and their healthy first-degree relatives.

AIMS: The gut microbiome has been recognized as a significant contributor to primary hepatocellular carcinoma (HCC), with mounting evidence indicating associations between bacterial components and cancers of the digestive system. METHODS AND RESULTS: Here, to characterize gut bacterial signature in patients with primary HCC and to assess the diagnostic potential of bacterial taxa for primary HCC, 21 HCC patients and 21 healthy first-degree relatives (control group) were enrolled in this study. Bacterial DNA in the fecal samples was quantified by 16S rRNA gene sequencing. We found that 743 operational taxonomic units (OTUs) were shared between patients with primary HCC and healthy controls. Of these, 197 OTUs were unique to patients with primary HCC, while 95 OTUs were unique to healthy subjects. Additionally, we observed significant differences in the abundance of Ruminococcaceae_UCG-014 and Romboutsia between patients with primary HCC and their healthy first-degree relatives. Besides, the relative abundance of Ruminococcaceae_UCG-014 and Prevotella_9 was positively correlated with physiological indicators including AST, ALT, ALB, or TBIL. Signature bacterial taxa could serve as non-invasive biomarkers, of which Romboutsia and Veillonella were identified as differential taxa in fecal samples from patients with HCC compared to healthy controls. Romboutsia showed a strong association with HCC (AUC = 0.802). Additionally, the combination of Romboutsia and Veillonella (AUC = 0.812) or the grouping of Fusobacterium, Faccalibacterium, and Peptostreptococcacae together (AUC = 0.762) exhibited promising outcomes for the diagnosis of HCC. CONCLUSIONS: The composition of gut microbes in patients with HCC was found to be significantly altered. Differential taxa Romboutsia, Veillonella, and Peptostreptococcacae could be tested for identification of HCC.

Lactic acid bacteria-derived exopolysaccharide: Formation, immunomodulatory ability, health effects, and structure-function relationship.

Exopolysaccharides (EPSs) synthesized by lactic acid bacteria (LAB) have implications for host health and act as food ingredients. Due to the variability of LAB-EPS (lactic acid bacteria-derived exopolysaccharide) gene clusters, especially the glycosyltransferase genes that determine monosaccharide composition, the structure of EPS is very rich. EPSs are synthesized by LAB through the extracellular synthesis pathway and the Wzx/Wzy-dependent pathway. LAB-EPS has a strong immunomodulatory ability. The EPSs produced by different genera of LAB, especially Lactobacillus, Leuconostoc, and Streptococcus, have different immunomodulatory abilities because of their specific structures. LAB-EPS possesses other health effects, including antitumor, antioxidant, intestinal barrier repair, antimicrobial, antiviral, and cholesterol-lowering activities. The bioactivities of LAB-EPS are tightly related to their structures such us monosaccharide composition, glycosidic bonds, and molecular weight (MW). For the excellent physicochemical property, LAB-EPS acts as product improvers in dairy, bakery food, and meat in terms of stability, emulsification, thickening, and gelling. We systematically summarize the detailed process of EPS from synthesis to application, with emphasis on physiological mechanisms of EPS, and specific structure-function relationship, which provides theoretical support for the potential commercial value in the pharmaceutical, chemical, food, and cosmetic industries.

Relationship of Gene-Structure-Antioxidant Ability of Exopolysaccharides Derived from Lactic Acid Bacteria: A Review.

Polysaccharides derived from lactic acid bacteria (LAB) have widespread industrial applications owing to their excellent safety profile and numerous biological properties. The antioxidant activity of exopolysaccharides (EPS) offers defense against disease conditions caused by oxidative stress. Several genes and gene clusters are involved in the biosynthesis of EPS and the determination of their structures, which play an important role in modulating their antioxidant ability. Under conditions of oxidative stress, EPS are involved in the activation of the nonenzyme (Keap1-Nrf2-ARE) response pathway and enzyme antioxidant system. The antioxidant activity of EPS is further enhanced by the targeted alteration of their structures, as well as by chemical methods. Enzymatic modification is the most commonly used method, though physical and biomolecular methods are also frequently used. A detailed summary of the biosynthetic processes, antioxidant mechanisms, and modifications of LAB-derived EPS is presented in this paper, and their gene-structure-function relationship has also been explored.

Lutispora saccharofermentans sp. nov., a mesophilic, non-spore-forming bacterium isolated from a lab-scale methanogenic landfill bioreactor digesting anaerobic sludge, and emendation of the genus Lutispora to include species which are non-spore-forming and mesophilic.

A novel anaerobic, mesophilic, non-spore-forming bacterium (strain m25T) was isolated from methanogenic enrichment cultures obtained from a lab-scale methanogenic landfill bioreactor containing anaerobic digester sludge. Cells were Gram-stain-negative, catalase-positive, oxidase-negative, rod-shaped, and motile by means of a flagellum. The genomic DNA G+C content was 40.11 mol%. The optimal NaCl concentration, temperature and pH for growth were 2.5 g l-1, 35 °C and at pH 7.0, respectively. Strain m25T was able to grow in the absence of yeast extract on glycerol, pyruvate, arginine and cysteine. In the presence of 0.2 % yeast extract, strain m25T grew on carbohydrates and was able to use glucose, cellobiose, fructose, raffinose and galactose. The novel strain could utilize glycerol, urea, pyruvate, peptone and tryptone. The major fatty acids were iso-C15  :  0, C14  :  0, C16  :  0 DMA (dimethyl acetal) and iso-C15 : 0 DMA. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate was closely related to Lutispora thermophila EBR46T (95.02 % 16S rRNA gene sequence similarity). Genome relatedness was determined using both average nucleotide identity and amino acid identity analyses, the results of which both strongly supported that strain m25T belongs to the genus Lutispora. Based on its unique phylogenetic features, strain m25T is considered to represent a novel species within the genus Lutispora. Moreover, based on its unique physiologic features, mainly the lack of spore formation, a proposal to amend the genus Lutispora is also provided to include the non-spore-forming and mesophilic species. Lutispora saccharofermentans sp. nov. is proposed. The type strain of the species is m25T (=DSM 112749T=ATCC TSD-268T).

Starter culture-related changes in free amino acids, biogenic amines profile, and antioxidant properties of fermented red beetroot grown in Poland.

Fermentation of two red beet cultivars (Wodan and Alto) with single-strain starter cultures consisting of selected strains of lactic acid bacteria (LAB) of plant origin (Weissella cibaria KKP2058, Levilactobacillus brevis ZF165) and a multi-strain culture (containing W. cibaria KKP2058, L. brevis ZF165, Lactiplantibacillus plantarum KKP1822, Limosilactobacillus fermentum KKP1820, and Leuconostoc mesenteroides JEIIF) was performed to evaluate their impact on betalains, free amino acids, formation of biogenic amines, and antioxidative properties of the juice formed. Next-generation sequencing data analysis used to identify the microbial community revealed that the starter cultures promoted the dominance of the genus Lactobacillus, and decreased the proportion of spoilage bacteria compared to spontaneously fermented juices. Generally, the fermentation process significantly influenced the amount of the analyzed compounds, leading in most cases to their reduction. The observed changes in the studied parameters depended on the starter culture used, indicating different metabolic activities of the LAB strains towards bioactive compounds. The use of multi-strain starter cultures allowed to largely prevent the reduction of betacyanins and histamine formation.

A novel species of lactic acid bacteria, Ligilactobacillus pabuli sp. nov., isolated from alfalfa silage.

In this study, we isolated a novel strain of lactic acid bacteria, AF129T, from alfalfa silage prepared locally in Morioka, Iwate, Japan. Polyphasic taxonomy was used to characterize the bacterial strain. The bacterium was rod-shaped, Gram-stain-positive, non-spore-forming and catalase-negative. The strain grew at various temperatures (15-40°C) and pH levels (4.0-8.0). The optimum growth conditions were a temperature of 30°C and a pH of 6.0. AF129T exhibited growth at salt (NaCl) concentrations of up to 6.5 % (w/v). The G+C content of the strain's genomic DNA was 41.5 %. The major fatty acids were C16 : 0, C18 : 1ω9c, C19 : 0cyclo ω8c and summed feature 8. 16S rRNA gene sequencing revealed that AF129T represents a member of the genus Ligilactobacillus and it has higher sequence similarities with Ligilactobacillus pobuzihii (98.4 %), Ligilactobacillus acidipiscis (97.5 %) and Ligilactobacillus salitolerans (97.4 %). The digital DNA-DNA hybridization values for AF129T and phylogenetically related species of the genus Ligilactobacillus ranged from 19.8% to 24.1%. The average nucleotide identity of the strain with its closely related taxa was lower than the threshold (95 %-96 %) used for species differentiation. In the light of the above-mentioned physiological, genotypic, chemotaxonomic and phylogenetic evidence, we confirm that AF129T represents a member of the genus Ligilactobacillus and constitutes a novel species; we propose the name Ligilactobacillus pabuli sp. nov. for this species. The type strain is AF129T =MAFF 518002T =JCM 34518T=BCRC 81335T.

Growth studies of dominant lactic acid bacteria in orange juice and selection of strains to ferment citric fruit juices with probiotic potential.

The study aimed to evaluate the ability of dominant lactic acid bacteria (LAB) in orange juice to growth on N-depleted MRS medium supplemented or not with cysteine (mMRS), then to select the most nutritionally promising strains for growth assays in the food matrix and evaluation of beneficial attributes for fruit juice fermentation. Levilactobacillus brevis and Lactiplantibacillus plantarum were dominant species among the total of 103 LAB isolates as confirmed by multiplex PCR and/or 16 s rDNA sequence analysis. Based on growing lower than 20% and higher than 70% in mMRS (1.0 g/l meat extract, without peptone and yeast extract) with and without cysteine requirement, one L. brevis (JNB23) and two L. plantarum (JNB21 and JNB25) were selected. These bacteria and the L. plantarum strains N4 and N8 (previously isolated from oranges peel) when inoculated in orange juice grew up to 1.0 log cfu/ml for 24 h incubation at 30 °C and mainly produced lactic acid, with strains JNB25 and JNB23 reaching the highest and lowest cell densities in agreement with their nutritional exigency. In addition, all L. plantarum strains exhibited antagonistic activity against the majority of tested bacterial pathogens (in opposition to L. brevis), ability to grow or survive to pH 3.0 for 3 h, to grow with 0.5% sodium taurocholate, and a decrease after simulated gastrointestinal digestion assay which did not exceed 1.0 or 2.0 log units, depending on the strain. Thus, autochthonous L. plantarum strains with ability for overcoming nutritional limitations and beneficial attributes are promising candidates for further investigations as novel probiotic and/or preservative starters to ferment citric fruit juices.

Predicting Inchinkoto efficacy, in patients with obstructive jaundice associated with malignant tumors, through pharmacomicrobiomics.

Inchinkoto (ICKT) is a popular choleretic and hepatoprotective herbal medicine that is widely used in Japan. Geniposide, a major ingredient of ICKT, is metabolized to genipin by gut microbiota, which exerts a choleretic effect. This study investigates the relationship between stool genipin-producing activity and diversity of the clinical effect of ICKT in patients with malignant obstructive jaundice. Fifty-two patients with malignant obstructive jaundice who underwent external biliary drainage were included. ICKT was administered as three packets per day (7.5 g/day) for three days and 2.5 g on the morning of the fourth day. Stool samples were collected before ICKT administration and bile flow was monitored on a daily basis. The microbiome, genipin-producing activity, and organic acids in stools were analyzed. The Shannon-Wiener (SW) index was calculated to evaluate gut microbiome diversity. The stool genipin-producing activity showed a significant positive correlation with the SW index. Stool genipin-producing activity positively correlated with the order Clostridia (obligate anaerobes), but negatively correlated with the order Lactobacillales (facultative anaerobes). Moreover, stool genipin-producing activity was positively correlated to the concentration valeric acid, but negatively correlated to the concentration of lactic acid and succinic acid. The change of bile flow at 2 and 3 days after ICKT administration showed significant positive correlation with genipin-producing activity (correlation coefficient, 0.40 and 0.29, respectively, P < 0.05). An analysis of stool profile, including stool genipin-producing activity, may predict the efficacy of ICKT. Modification of the microbiome may be a target to enhance the therapeutic effect of ICKT.

Screening of Lactic Acid Bacterial Strains with Antiviral Activity Against Porcine Epidemic Diarrhea.

Newly emerging and re-emerging viral infectious diseases cause significant economic losses in swine production. Efficacious vaccines have not yet been developed for several major swine infectious diseases, including porcine epidemic diarrhea virus (PEDV). We used the PEDV-infected Vero cell model to screen lactic acid bacteria (LAB) strains with antiviral activity. Sixty LAB strains were isolated from the feces of nursing piglets. After the elimination of LAB strains with high cytotoxicity to Vero cells, the protective effects of the remaining 6 strains against PEDV infection were determined. Vero cells pretreated with the intracellular extracts or cell wall fractions of YM22 and YM33 strains for 24 h before infection with PEDV showed significantly higher cell viabilities and lower mRNA expression of PEDV nucleocapsid (PEDV-N) than the unpretreated cells, indicating that the intracellular extracts and cell wall fractions of YM22 and YM33 possessed prophylactic effects on Vero cells against PEDV infection. PEDV-infection significantly increased the mRNA expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) in Vero cells. However, pretreatment of Vero cells with the cell wall fractions of YM22 and YM33 decreased the mRNA expression of TNF-α and IL-8, which could be a mechanism associated with the protective effects of YM22 and YM33 against PEDV. Based on the biochemical characteristics and phylogenetic analyses, YM22 and YM33 were identified as Ligilactobacillus agilis (basonym: Lactobacillus agilis) and Ligilactobacillus salivarius (basonym: Lactobacillus salivarius), respectively. These findings suggest that L. agilis YM22 and L. salivarius YM33 could provide some levels of protective effects against PEDV infections.

Using PacBio sequencing to investigate the effects of treatment with lactic acid bacteria or antibiotics on cow endometritis

BACKGROUND: Endometritis is the most common disease of dairy cows and traditionally treated with antibiotics. Lactic acid bacteria can inhibit the growth of pathogens and also have potential for treatment of endometritis. Using PacBio single-molecule real-time sequencing technology, we sequenced the fulllength l6S rRNA of the microbiota in uterine mucus samples from 31 cows with endometritis, treated with lactic acid bacteria (experimental [E] group) and antibiotics (control [C] group) separately. Microbiota profiles taken before and after treatment were compared. RESULTS: After both treatments, bacterial species richness was significantly higher than before, but there was no significant difference in bacterial diversity. Abundance of some bacteria increased after both lactic acid bacteria and antibiotic treatment: Lactobacillus helveticus, Lactococcus lactis, Lactococcus raffinolactis, Pseudomonas alcaligenes and Pseudomonas veronii. The bacterial species that significantly decreased in abundance varied depending on whether the cows had been treated with lactic acid bacteria or antibiotics. Abundance of Staphylococcus equorum and Treponema brennaborense increased after lactic acid bacteria treatment but decreased after antibiotic treatment. According to COG-based functional metagenomic predictions, 384 functional proteins were significantly differently expressed after treatment. E and C group protein expression pathways were significantly higher than before treatment (p < 0.05). CONCLUSIONS: In this study, we found that lactic acid bacteria could cure endometritis and restore a normal physiological state, while avoiding the disadvantages of antibiotic treatment, such as the reductions in abundance of beneficial microbiota. This suggests that lactic acid bacteria treatment has potential as an alternative to antibiotics in the treatment of endometritis in cattle.

The lactic acid bacteria and yeast community of home-made sauerkraut from three provinces in Southwest China.

The aim of this study was to investigate the lactic acid bacteria (LAB) and yeast community from home-made sauerkraut collected from Southwest China through culture-dependent and culture-independent technology. Forty-eight samples of home-made sauerkraut were collected from households at three different locations in Southwest China. The pH, total acidity and salt contents among these fermented vegetables were 3.69 ± 0.42, 0.86 ± 0.43 g/100 ml, and 3.86 ± 2.55 g/100 ml, respectively. The number of lactic acid bacteria (LAB) and yeasts were 7.25 ± 1.05 log10 colony-forming units (CFU)/ml and 3.74 ± 1.01 log CFU/ml, respectively. A total of 182 LAB and 81 yeast isolates were identified. The dominant isolates were Lactobacillus plantarum, L. brevis, Pediococcus ethanolidurans, Pichia membranifaciens, P. fermentans and Kazachstania bulderi. Denaturing gradient gel electrophoresis (DGGE) showed that L. plantarum, uncultured Lactobacillus sp, P. ethanolidurans, and K. exigua were the predominant microflora. Our studies demonstrated that the DGGE technique combined with a culture-dependent method is very effective for studying the LAB and yeast community in Chinese traditional fermentation vegetables. The results will give us an understanding of LAB and yeast community of Chinese sauerkraut and improve the knowledge of LAB and yeast community of Chinese sauerkraut.

Gas chromatography-mass spectrometry-based metabolomics analysis of metabolites in commercial and inoculated pickles.

BACKGROUND: Starter cultures are nowadays more and more used to make inoculated pickles (IPs), but it remains unclear whether there are differences in flavors between IPs and naturally fermented pickles. In this study 16 commercial pickles (CPs) produced by spontaneous fermentation method were purchased from markets in Sichuan province and Chongqing. Ten strains of three lactic acid bacteria species - Lactobacillus plantarum, Leuconostoc mesenteroides and Pediococcus ethanolidurans - were selected as single starter cultures to produce IPs. RESULTS: Differences in flavor components between the CPs and IPs were monitored using a combination of gas chromatography-mass spectrometry and multivariate statistical methods. Higher levels of nonvolatile substances such as glucose, fructose, tagatose, sucrose, lactic acid and mannitol were detected in most IPs than in the CPs. The values of flavor characteristics such as sweetness, umami and astringency, which were correlated positively with consumers' overall preferences for pickles, were higher in the IPs than in the CPs. Volatile compounds such as geranyl acetate, dimethyl trisulfide, eucalyptol and linalool were distinguished as the main compounds that contributed to the flavor characteristics of the CPs. In addition to dimethyl trisulfide, dimethyl disulfide was also an odor contributor to the IPs. CONCLUSIONS: The CPs and IPs had different flavor characteristics, especially in the composition and content of volatile components, and the inoculation method reflected some fermentation advantages, which could reduce the bitterness and increase umami and lead to a higher score of sensory preference. This will be helpful for industrial production. © 2020 Society of Chemical Industry.

Twenty years of research on HPV vaccines based on genetically modified lactic acid bacteria: an overview on the gut-vagina axis.

Most cervical cancer (CxCa) are related to persistent infection with high-risk human papillomavirus (HR-HPV) in the cervical mucosa, suggesting that an induction of mucosal cell-mediated immunity against HR-HPV oncoproteins can be a promising strategy to fight HPV-associated CxCa. From this perspective, many pre-clinical and clinical trials have proved the potential of lactic acid bacteria (LAB) genetically modified to deliver recombinant antigens to induce mucosal, humoral and cellular immunity in the host. Altogether, the outcomes of these studies suggest that there are several key factors to consider that may offer guidance on improvement protein yield and improving immune response. Overall, these findings showed that oral LAB-based mucosal HPV vaccines expressing inducible surface-anchored antigens display a higher potential to induce particularly specific systemic and mucosal cytotoxic cellular immune responses. In this review, we describe all LAB-based HPV vaccine investigations by reviewing databases from international studies between 2000 and 2020. Our aim is to promote the therapeutic HPV vaccines knowledge and to complete the gaps in this field to empower scientists worldwide to make proper decisions regarding the best strategies for the development of therapeutic HPV vaccines.

Oxidative stress tolerance and antioxidant capacity of lactic acid bacteria as probiotic: a systematic review.

Lactic acid bacteria (LAB) are the most frequently used probiotics in fermented foods and beverages and as food supplements for humans or animals, owing to their multiple beneficial features, which appear to be partially associated with their antioxidant properties. LAB can help improve food quality and flavor and prevent numerous disorders caused by oxidation in the host. In this review, we discuss the oxidative stress tolerance, the antioxidant capacity related herewith, and the underlying mechanisms and signaling pathways in probiotic LAB. In addition, we discuss appropriate methods used to evaluate the antioxidant capacity of probiotic LAB. The aim of the present review is to provide an overview of the current state of the research associated with the oxidative stress tolerance and antioxidant capacity of LAB.

Phylogenetic distribution of the bacteriocin repertoire of lactic acid bacteria species associated with artisanal cheese.

The microbiota contributes to artisanal cheese bioprotection and biopreservation through inter and intraspecific competition. This work aimed to investigate the phylogenetic distribution of the repertoire of bacteriocin structural genes of model lactic acid bacteria (LAB) in order to investigate its respective role in the artisanal cheeses microenvironment. A phylogenetic analysis of the rRNA 16S gene from 445 model strains of LAB was conducted using bayesian inference and the repertoire of bacteriocin genes was predicted from these strains by BAGEL software. Bacterial strains were clustered in five monophyletic clades (A, B, C, D and E) with high posterior probability values (PP > 0.99). One bacteriocin structural gene was predicted for 88.5% of the analyzed strains. The majority of the species encoded different classes of bacteriocins. Greater diversity of bacteriocin genes was found for strains included in clade A, comprising Lactococcus lactis, Streptococcus agalactiae, Streptococcus thermophilus, Streptococcus macedonicus, Enterococcus faecalis and Enterococcus faecium. In addition, Lactococcus lactis presented higher diversity of bacteriocin classes, encoding glycocins, lanthipeptides, sactipeptides, cyclic and linear azole-containing peptides, included in bacteriocins class I, besides class II and III. The results suggest that the distribution of bacteriocin structural genes is related to the phylogenetic clades of LAB species, with a higher frequency in some specific clades. Information comprised in this study contributes to comprehend the bacterial competition mechanisms in the artisanal cheese microenvironment.

Adhesion mechanisms of lactic acid bacteria: conventional and novel approaches for testing.

Adhesion ability is a primary criterion for the selection of probiotic microorganisms. Lactic acid bacteria contribute the majority of microorganisms with probiotic properties. They have several important mechanisms for intestinal epithelial cell adhesion. In order to adhere to the intestinal cells, they generally use various structures such as flagella, pili, S layer proteins, lipoteichoic acid, exopolysaccharides and mucus binding proteins. Various in vitro experiments were designed or study models were developed to reveal the mechanisms they utilize for binding to the intestinal cells, yet, the mechanisms for their adhesion are not fully explained. The major disadvantage of conventional models is the lack of layers forming the intestinal mucosa. Besides, these models omit the presence of natural microbiota, digestive conditions and the presence of a food matrix. Because of the disadvantages of existing models, natural tissues or novel applications like 3D organ cultures, which are better able to mimic in vivo conditions, are preferred.

Secretion and functional expression of Mycobacterium bovis antigens MPB70 and MPB83 in lactic acid bacteria.

The aim of this study was to determine the reliability of lactic acid bacteria (LAB) as heterologous hosts for the expression of MPB70 and MPB83, two Mycobacterium bovis antigens that possess diagnostics and immunogenic properties, respectively. We therefore generated recombinant cells of Lactococcus lactis and Lactobacillus plantarum that carried hybrid genes encoding MPB70 and MPB83 fused to signal peptides that are specifically recognized by LAB. Only L. lactis was able to secrete MPB70 using the L. lactis signal peptide Usp45, and to produce MPB83 as an immunogenic membrane protein following its expression with the signal peptide of the L. plantarum lipoprotein prsA. Inactivated cells of MPB83-expressing L. lactis cultures enhanced NF-κB activation in macrophages. Our results show that L. lactis is a reliable host for the secretion and functional expression of antigens that are naturally produced by M. bovis, the causative agent of bovine tuberculosis (bTB). This represents the first step on a long process to establishing whether recombinant LAB could serve as a food-grade platform for potential diagnostic tools and/or vaccine interventions for use against bTB, a chronic disease that primarily affects cattle but also humans and a wide range of domestic and wild animals.